title: Antagonistic roles of Dmrt1 and Foxl2 in sex differentiation via estrogen production in tilapia as demonstrated by TALENs.
abstract: Transcription activator-like effector nucleases (TALENs) are a powerful approach for targeted genome editing and have been proved to be effective in several organisms. In this study, we reported that TALENs can induce somatic mutations in Nile tilapia, an important species for worldwide aquaculture, with reliably high efficiency. Six pairs of TALENs were constructed to target genes related to sex differentiation, including dmrt1, foxl2, cyp19a1a, gsdf, igf3, and nrob1b, and all resulted in indel mutations with maximum efficiencies of up to 81% at the targeted loci. Effects of dmrt1 and foxl2 mutation on gonadal phenotype, sex differentiation, and related gene expression were analyzed by histology, immunohistochemistry, and real-time PCR. In Dmrt1-deficient testes, phenotypes of significant testicular regression, including deformed efferent ducts, degenerated spermatogonia or even a complete loss of germ cells, and proliferation of steroidogenic cells, were observed. In addition, disruption of Dmrt1 in XY fish resulted in increased foxl2 and cyp19a1a expression and serum estradiol-17β and 11-ketotestosterone levels. On the contrary, deficiency of Foxl2 in XX fish exhibited varying degrees of oocyte degeneration and significantly decreased aromatase gene expression and serum estradiol-17β levels. Some Foxl2-deficient fish even exhibited complete sex reversal with high expression of Dmrt1 and Cyp11b2. Furthermore, disruption of Cyp19a1a in XX fish led to partial sex reversal with Dmrt1 and Cyp11b2 expression. Taken together, our data demonstrated that TALENs are an effective tool for targeted gene editing in tilapia genome. Foxl2 and Dmrt1 play antagonistic roles in sex differentiation in Nile tilapia via regulating cyp19a1a expression and estrogen production.
authors.affiliation: Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Science, Southwest University, Chongqing, China. wdeshou@swu.edu.cn.
authors.forename: Ming-Hui
authors.initials: MH
authors.lastname: Li
authors.forename: Hui-Hui
authors.initials: HH
authors.lastname: Yang
authors.forename: Meng-Ru
authors.initials: MR
authors.lastname: Li
authors.forename: Yun-Lv
authors.initials: YL
authors.lastname: Sun
authors.forename: Xiao-Long
authors.initials: XL
authors.lastname: Jiang
authors.forename: Qing-Ping
authors.initials: QP
authors.lastname: Xie
authors.forename: Ting-Ru
authors.initials: TR
authors.lastname: Wang
authors.forename: Hong-Juan
authors.initials: HJ
authors.lastname: Shi
authors.forename: Li-Na
authors.initials: LN
authors.lastname: Sun
authors.forename: Lin-Yan
authors.initials: LY
authors.lastname: Zhou
authors.forename: De-Shou
authors.initials: DS
authors.lastname: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism; D004967:Estrogens / Q000235:genetics / Q000378:metabolism*; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D009154:Mutation; D012733:Sex Differentiation / Q000502:physiology*; D017210:Tilapia / Q000196:embryology / Q000254:growth & development* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: C110547:DMRT1 protein; D004967:Estrogens; D029941:Fish Proteins; D051858:Forkhead Transcription Factors; D014157:Transcription Factors; D003851:Deoxyribonucleases